Aggressive behavior is a damaging influence in our society, since by definition it is intended to inflict physical or psychological harm. In humans, growing up in a socially adverse environment can strongly influence the development of aggressive tendencies, and genotypic variation can also predispose individuals to aggression. DNA methylation is an epigenetic modification that exists in relatively stable patterns, but can change at specific loci in response to the environment. This stable/dynamic duality potentially places DNA methylation at the regulatory interface between genotypic and environmental influences on behavior. This proposal will investigate whether DNA methylation mediates the influences of genotype and environment on aggression in the honey bee, a model that exhibits well-characterized aggressive behaviors, and unlike the fruit fly, has a fully functional, mammalian-like methylation system. Strains of bees differ in their aggressiveness, and our prior work has shown that the highly aggressive African honey bee (AHB) shows differences in brain gene expression and DNA methylation from the less aggressive European honey bee (EHB). In addition, EHB provoked to aggression by exposure to alarm pheromone show expression changes in some of the same genes. We will test the hypothesis that DNA methylation serves as a stable epigenetic mark regulating inherited differences in aggression, while also acting as a dynamic regulator responding to environmental stimuli that promote aggression. In mammals, DNA methylation patterns can be modulated through active DNA demethylation, which is mediated by ten-eleven translocase (TET) and thymine DNA glycosylase (TDG) enzymes. To determine whether a TET/TDG-dependent DNA demethylation mechanism exists in bees and regulates aggression, TDG will be knocked down in the bee brain using RNA interference (RNAi); preliminary results indicate a ca. 20% knockdown. We will measure DNA demethylation and aggression in the TDG knockdown bees, as well as the accumulation of oxidized 5-methylcytosine derivatives, which are known intermediates of active DNA methylation in other organisms. To explore whether DNA methylation also establishes aggression as a stable trait associated with genotype, we will examine the stability of DNA methylation patterns at aggression-related genes in AHB and EHB. Lastly, we will compare methylation patterns at aggression-related genes between the hereditary and environmentally-induced aggressive contexts. We expect to observe similar methylation patterns between the two, supporting our hypothesis that DNA methylation is a regulator underlying both genotypic and environmental effects on aggression.